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Depletion of neuropeptides in rat parotid glands and declining atropine-resistant salivary secretion upon continuous parasympathetic nerve stimulation
Regulatory Peptides, 11 (1985) 353-359
353
Elsevier RPT 00396
Depletion of neuropeptides in rat parotid glands and declining atropine-resistant salivary secretion upon continuous parasympathetic nerve stimulation J. Ekstr6m a, E. Brodin d, R. E k m a n b, R. Hfikanson c, B. M~msson a and G. Tobin a Departments of'Physiology, bNeurochemistry and CPharmacology, University of Lund, Lund, and dDepartment of Pharmacology, Karolinska lnstitutet, Stockholm, Sweden (Received 3 May 1985; accepted for publication 19 May 1985)
Summary In rats the parasympathetic auriculo-temporal nerve on one side was continuously stimulated at 40 Hz for 20-80 min in the presence of adrenergic blockers (dihydroergotamine and propranolol) + / - atropine. During the first 10 min this gave rise to a flow of saliva from the parotid gland that in the atropinized rats amounted to 35% of that found in rats not treated with atropine, while the protein and amylase outputs were 75% of those in non-atropinized rats. The atropine-resistant secretion of fluid and proteins declined to 5-10% of the initial value within 40 min but did not cease completely even after 80 min. The marked reduction in secretory responses was not due to desensitization or exhaustion of the gland cells. The nerve stimulation reduced the parotid gland content of vasoactive intestinal peptide (VIP) and substance P (SP) to approximately 60 and 25% of that of contralateral glands after 20 and 60 min, respectively. The probable explanation for the decline in secretory response seems to be depletion of non-adrenergic, non-cholinergic transmitter(s). The present results suggest that neuropeptides are involved in the regulation of salivary secretion but provide no direct evidence that either VIP or SP is responsible for the atropine-resistant salivary secretion. vasoactive intestinal peptide; substance P; muscarinic block; auriculo-temporal nerve stimulation; fluid, amylase and protein secretion
Address all correspondence to." Dr. J. Ekstr6m, Dept. of Physiology, University of Lund, S61vegatan 10, S-223 62 Lund, Sweden. 0167-0115/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
354
Introduction
Salivary secretion from rat parotid gland is evoked not only by cholinergic and adrenergic agents [1] but also by neuropeptides. Substance P (SP) injected intravenously causes a lively flow of saliva [2]. Vasoactive intestinal peptide (VIP), on the other hand, causes the gland to secrete a small amount of protein-rich saliva [3]. SPand VIP-containing nerve fibres reach the parotid gland via the parasympathetic auriculo-temporal nerve; the total amounts of these peptides in the gland are reduced by about 95% following parasympathetic denervation [4]. Interestingly, even in the presence of atropine and ~- and ~-adrenoceptor blockers electrical stimulation of the auriculo-temporal nerve evokes a secretion of saliva in the rat, which amounts to 5-10% of that obtained upon nerve stimulation before administration of atropine [5]. This atropine-resistant secretion, rich in proteins, is not observed at stimulation frequencies below 5 Hz; the maximal flow rate occurs at 40 Hz [6]. In the present study an attempt was made to deplete the neuronal stores of SP and VIP in the parotid gland by continuous stimulation of the auriculo-temporal nerve. Further, it was investigated whether this protocol would affect the atropine-resistant flow of saliva and output of amylase and proteins.
Methods
48 adult male Sprague-Dawley rats, weighing 303 + 5 g (mean ± S.E.M.) were anaesthetized with chloralose (100 mg/kg) through a femoral venous cannula after induction with diethyl ether. They were provided with a tracheal cannula and a rectal thermometer; the body temperature was kept at about 37.5"C. The duct of the parotid gland was exposed near its entrance in the mouth and cannulated with a glass cannula. The auriculo-temporal nerve, carrying the parasympathetic nerve fibres to the gland [7,8], was exposed medially to the mandible and the nerve was cut where it emerged from the base of the skull. To exclude adrenergic effects the ct-adrenoceptor blocker dihydroergotamine (0.5 mg/kg i.v.) and the fl-adrenoceptor blocker propranolol (0.5 mg/kg i.v.) were given to all the rats. The peripheral end of the nerve was stimulated with supramaximal voltage, 6 V, and at a duration of 2 ms, at a frequency of 40 Hz using a bipolar electrode and a Grass $48 stimulator. Saliva was collected from the cannula in small ice-cooled tubes; the tubes were changed every 10 min. The saliva collected during each 10 min period was weighed and frozen at - 2 0 ° C until analysed for protein and amylase. Protein was determined by the method of Lowry et al. [9] using bovine serum albumin as standard. Amylase was determined colorimetrically using ~-4-nitrophenyl maltoheptaoside (Boehringer Mannheim) as substrate [10]. At the end of the experiment both the stimulated and the non-stimulated parotid gland were removed, weighed, immediately frozen and stored ,at - 70°C until extracted for determination of either VIP or SP. For the assay of VIP-like immunoreactivity (below referred to as VIP), the specimens were extracted in 1-2 ml boiling 0.5 M acetic acid for 10 min, homogenized and centrifuged at 3000 x g for 10 min at room temperature. The supernatants were
355 freeze-dried. The samples were reconstituted in 0.05 M sodium phosphate buffer (pH 7.4) containing 0.5% human serum albumin (Behringwerke, Marburg/Lahn, F.R.G.) and 0.1 M NaC1. Immunoreactive VIP was quantitated using a rabbit antiserum to VIP (No. 7852, Milab, Malmr, Sweden) and radioiodinated VIP [11]; 200 #1 of antiserum (diluted 1 : 16 000) were incubated first with 500/~1 of standard or tissue extract for 3 days at 4"C and then with 200/A (about 4000 cpm) of 125I-VIP for another 2 days at 4°C. Bound and free 12sI-VIP were separated using a second antibody. Each tissue extract was assayed in serial dilution. At times the extracts gave dilution curves that were parallel with the standard VIP curve in the high dilution range only, suggesting the presence of a VIP-like peptide rather than authentic VIP. This was the case when the VIP concentrations in the parotid glands were low. The standard used was synthetic porcine VIP, purchased from Peninsula (Belmont, CA). The concentrations of VIP in the parotid gland extracts were assessed in the high dilution range where the curve was parallel to the standard curve and expressed as equivalents of porcine VIP. It should be recognized that for the reasons specified the absolute values given may be subject to error. For the assay of SP-like immunoreactivity (below referred to as SP), the specimens were minced while still frozen and extracted in 10 volumes of 1.0 M acetic acid in a boiling water bath for 10 min. Following homogenization and centrifugation the supernatants were freeze-dried and redissolved in assay buffer immediately prior to radioimmunoassay. Antiserum K-25 which is directed against the C-terminus of SP was used in a final dilution ot 1:350000, and 12SI-TyrS-Sp served as tracer [12]. The extracts were assayed in duplicate at 3 dilutions. Dilution curves of salivary gland extracts were parallel with the SP standard curve. The levels of VIP and SP were expressed in terms of total amounts, pmol/gland. The drugs used in the studies on secretory responses included atropine sulphate (Sigma), dihydroergotamine methansulphonate (Sandoz), propranolol hydrochloride (ICI) and SP (Sigma). All drugs were injected intravenously. Student's t-test for unpaired or paired data when appropriate was used for estimation of statistical significances. Values are given as means + S.E.M.
Results
Gland weights After continuous stimulation for 20-80 min a periglandular oedema had developed both in the presence and in the absence of atropine. In 35 rats given atropine the wet weight of the parotid glands on the stimulated side was 240 + 6 mg and on the contralateral side 209 + 6 mg (P < 0.001). In 9 animals where the nerve had been stimulated in the absence of atropine the weight of the glands on the stimulated side was 224 q- 9 mg and on the contralateral side 203 + 8 mg (P < 0.01).
Secretory responses There was no spontaneous secretion from the parotid glands. Stimulation of the auriculo-temporal nerve (40 Hz) in non-atropinized animals resulted in salivary flow
356
within 1-2 s. Following treatment with atropine (2 mg/kg i.v.) the secretory response was not apparent until after about 10 s of stimulation. The atropine-resistant secretion during the first 10 min amounted to 231 + 9 mg (n=35) saliva. The output of amylase was 388 + 44 U (n=35) (Fig. 1), and of total protein 881 4- 86 #g (n=35). In the absence of atropine the amount of saliva secreted during the same 10 rain period was 635 + 37 mg (n=9). The output of amylase was 510 + 157 U (n=6) (Fig. 1), and of protein 1215 + 202 gg (n=6). Compared to non-atropinized rats, the amount of saliva, but not that of amylase and protein, secreted during these 10 min was significantly lower (P < 0.001) in atropinized rats. The amounts of saliva, amylase and protein secreted by each non-atropinized and atropinized rat during the initial 10 min period were all set to 100%. The values obtained in the following periods of 10 min were expressed as percentages. In the presence of atropine the flow rate declined markedly with time (Fig. 2). In the second period of observation (10-20 min) the amount of saliva secreted was reduced to 43% (n = 35); it was 13 (n = 26) and 6% (n = 17) in the fourth (30-40 min) and sixth period (50-60 min), respectively. No recovery in secretory responses was noted when stimulation was resumed half an hour after the end of an 80 min period of stimulation. In the absence of atropine the salivary flow rate declined somewhat initially, but remained at a fairly steady level thereafter (Fig. 2). The amount of saliva secreted in the second period was 83% (n = 9); it was 71% (n---5) in the sixth period. Also the output of amylase declined rapidly in the presence of atropine (Fig. 2). It was 28% (n = 35) in the second period, and 6 (n = 19) and 3% (n = 7) in the fourth and sixth period, respectively. In the absence of atropine the decline in amylase output was also great. The figures were 59 (n= 6), 34 (n= 6) and 27% (n= 5) in the second, fourth and sixth period, respectively (Fig. 2). The output of protein in the presence of atropine was 34 + 3% (n= 35) in the second period and 13 + 1% (n = 19) in the fourth period. In the absence of atropine
-ATROPINE +ATROPINE SALIVA AMYLASE 7001 6 600 5001
0 ] ]-
Fig. I. Secretion of saliva (in mg) and output of amylase (in U) from parotid glands during an initial 10-min period of electrical stimulation of the auriculo-temporal nerve (40 Hz, 6 V, 2 ms) in atropinized (2 mg/kg i.v.) and non-atropinized rats. Dihydroergotamine (0.5 mg/kg i.v.) and propranolol (0.5 mg/kg i.v.) were given to all animals. Values are means + S.E.M. Number of observations is indicated.
357 ~ ] -ATROPINE B +ATRORNE SALIVA %100-
4oi
AMYLASE %100
L0666 544
sol B
6o i
e
666
2o I
ol 0
20 40 60 80 min
0
20 40 60 80
min
Fig. 2. Secretion of saliva and output of amylase from parotid glands during 80 min of electrical stimulation of the auriculo-temporal nerve (40 Hz, 6 V, 2 ms) in atropinized and non-atropinized rats. Dihydroergotamine and propranolol were given to all animals (see Fig. 1). Saliva was collected in periods of 10 rain, weighed and analysed. The amounts of saliva and amylase secreted in each rat (both atropinized and non-atropinized) during the first 10-min period were set to 100% (see Fig. 1). All subsequent values were expressed as percentages. Means + S.E.M. Number of observations is indicated.
the protein output was 64 4- 13% (n=6) in the second period and 36 4- 10% (n = 5) in the fourth period. To test the secretory capacity of glands subjected to the prolonged stimulation in the atropinized animals, SP (0.5 #g/kg i.v.) was injected before and 3-5 min after 20-80 min of nerve stimulation. The response to SP after the period of stimulation was found to be larger (by 170%, n--14, P < 0.001) than that obtained before stimulation.
Parotid gland content of VIP and SP The total amount of VIP in 9 non-stimulated parotid glands was 3.4 4- 0.4 pmol, while that of SP in another series of 8 non-stimulated parotid glands was 859 4- 60 pmol. Stimulation of the auriculo-temporal nerve (at 40 Hz, in the presence of atropine) reduced the peptide content. After 20, 40 and 60 min of stimulation the amount of VIP in the stimulated glands, in % of that of contralateral glands, was 63 4- 16 (n = 3), 25 4- 3 (n = 4) and 25 (n = 2; 24, 26), respectively, while the corresponding figures for SP was 56 4- 7 (n = 3), 26 4- 1 (n = 3) and 23 (n = 2; 18, 28).
Discussion
The atropine resistant parotid secretion of fluid and protein, including amylase, decreased to about 5-10% of the initial values within 40 min stimulation of the auriculo-temporal nerve at 40 Hz. The secretion did not cease completely although the stimulation continued for another 40 min. A rest for half an hour was not enough to allow for a recovery in the secretory response. The marked reduction in secretion of water and proteins was not due to a general desensitization of the gland cells, nor was it due to exhaustion of the secretory machinery: the secretory response to SP injected intravenously was greater after than before nerve stimulation and
358
further, in rats not given atropine the salivary flow rate and output of protein, and amylase, exceeded that observed in the atropine treated rats. The probable explanation for the rapid decline of the secretory response in the presence of atropine on stimulation of the auriculo-temporal nerve seems to be depletion of non-adrenergic, non-cholinergic transmitter(s). Both VIP and SP would fit into that role, since the parotid gland content of both these peptides was markedly reduced in approximate parallel to the decline in the secretory response. After 40 min of nerve stimulation the total amounts of VIP and SP were reduced to 25%. There was no further decrease during the following 20 min. Interestingly, in the cat dental pulp complete depletion of SP did not occur following long-lasting antidromic nervestimulation [13]. Prolonged nerve stimulation probably evokes a rapid decrease in all neuropeptides in the parotid gland. A depletion can be anticipated since the neuropeptides are synthesized in the cell body of the neurone. Replenishment of the stores in the periphery of the neurone requires axonal transport of preformed, packaged peptides [14]. The present results strongly suggest the involvement of peptides in the regulation of salivary secretion but provide no direct evidence that either SP or VIP is responsible for the atropine-resistant secretion. Since both peptides, injected into the blood stream, stimulate salivary secretion this seems highly likely, however. Interestingly, injection of SP during a period of ongoing stimulation of the auriculo-temporal nerve was recently shown to potentiate the atropine-resistant salivary secretion. Furthermore, the secretory effect of VIP was greatly enhanced by SP (Ekstrfm and Olgart, unpublished observations). Secretion of total protein and amylase seems to a large extent to depend on nonadrenergic, non-cholinergic transmission. In the atropine treated rats total outputs of protein and amylase during the initial 10-min period of nerve stimulation were in both cases 75% of those in untreated rats, whereas the fluid secretion was 35% of controls. These figures are relatively high compared to those earlier reported [5,6]. It is likely that the atropine-resistant secretion has been underestimated previously, since in earlier studies the experimental protocol was to test frequency-response relations before and after atropine in one and the same animal. If the stimulation has been allowed to continue over several minutes at high frequencies the non-adrenergic, non-cholinergic transmitter is probably already partially depleted when the stimulation is repeated in the presence of atropine. Hence, it is probable that atropineresistant salivary secretion will be possible to demonstrate in other species too.
Acknowledgements This study was supported by grants from the Swedish Medical Research Council (14X-05927, 14X-6836, 04X-1007), the Faculties of Medicine and Dentistry, University of Lund, and Dr. P. H~tkansson's Foundation.
359
References 1 Ekstr6m, J., Sensitization of the rat parotid gland to secretagogues following either parasympathetic denervation or sympathetic denervation or decentralization, Acta Physiol. Scand., 108 (1980) 253-261. 2 Ekstr6m, J. and Wahlestedt, C., Supersensitivity to substance P and physalaemin in rat salivary glands after denervation or decentralization, Acta Physiol. Scand., 115 (1982) 437--446. 3 Ekstr6m, J., Milnsson, B. and Tobin, G., Vasoactive intestinal peptide evoked secretion of fluid and protein from rat salivary glands and the development of supersensitivity, Acta Physiol. Scand., 119 (1983) 169-175. 4 Ekstr6m, J., Brodin, E., Ekman, R., H~tkanson, R. and Sundler, F., Vasoactive intestinal peptide and substance P in salivary glands of the rat following denervation or duct ligation, Regul. Peptides, 10 (1984) I-I0. 5 Ekstr6m, J., M~nsson, B., Tobin, G., Garrett, J.R. and Thulin, A., Atropine-resistant secretion of parotid saliva on stimulation of the auriculotemporal nerve in the rat, Acta Physiol. Scand., 119 (1983) 445--449. 6 Ekstr6m, J., M~nsson, B. and Tobin, G., Effects of atropine or high frequency burst excitation on the composition of parasympathetic rat parotid saliva, Acta Physiol. Stand., 122 (1984) 409--414. 7 Ekstr6m, J., Choline acetyltransferase activity in rat salivary glands after cellulose rich diet or treatment with an atropine-like drug, Quart. J. Exp. Physiol., 59 (1974) 191-199. 8 Aim, P. and Ekstr6m, J., Cholinergic nerves of unknown origin in the parotid glands of rats, Arch. Oral Biol., 21 (1976) 417--421. 9 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. I0 H~igele, E.-O., Schaich, E., Rauscher, E., Lehmann, P., Burk, H. and Wahlefeld, A.-W., Mechanism of action of human pancreatic and salivary ct-4-nitrophenyl maltoheptaoside substrate, Clin. Chem., 28/11 (1982) 2201-2205. 11 Fahrenkrug, J. and Schaffalitzky de Muckadell, O.B., Radioimmunoassay of vasoactive intestinal polypeptide (VII)) in plasma, J. Lab. Clin. Med., 89 (1977) 1379-1387. 12 Brodin, E., Nilsson, G. and Folkers, K., Characterisation of two substance P anti,era, Acta Physiol. Scand., 114 (1982) 53-57. 13 Gazelius, B., Brodin, E. and Olgart, L., Depletion of substance P-like immunoreactivity in the cat dental pulp by antidromic nerve stimulation, Acta Physiol. Scand., 111 (1981) 319-327. 14 H~tkanson, R. and Sundler, F., The design of the neuroendocrine system: a unifying concept and its consequences, Trends Pharmacol. Sci., 4 (1983) 41-44.
353
Elsevier RPT 00396
Depletion of neuropeptides in rat parotid glands and declining atropine-resistant salivary secretion upon continuous parasympathetic nerve stimulation J. Ekstr6m a, E. Brodin d, R. E k m a n b, R. Hfikanson c, B. M~msson a and G. Tobin a Departments of'Physiology, bNeurochemistry and CPharmacology, University of Lund, Lund, and dDepartment of Pharmacology, Karolinska lnstitutet, Stockholm, Sweden (Received 3 May 1985; accepted for publication 19 May 1985)
Summary In rats the parasympathetic auriculo-temporal nerve on one side was continuously stimulated at 40 Hz for 20-80 min in the presence of adrenergic blockers (dihydroergotamine and propranolol) + / - atropine. During the first 10 min this gave rise to a flow of saliva from the parotid gland that in the atropinized rats amounted to 35% of that found in rats not treated with atropine, while the protein and amylase outputs were 75% of those in non-atropinized rats. The atropine-resistant secretion of fluid and proteins declined to 5-10% of the initial value within 40 min but did not cease completely even after 80 min. The marked reduction in secretory responses was not due to desensitization or exhaustion of the gland cells. The nerve stimulation reduced the parotid gland content of vasoactive intestinal peptide (VIP) and substance P (SP) to approximately 60 and 25% of that of contralateral glands after 20 and 60 min, respectively. The probable explanation for the decline in secretory response seems to be depletion of non-adrenergic, non-cholinergic transmitter(s). The present results suggest that neuropeptides are involved in the regulation of salivary secretion but provide no direct evidence that either VIP or SP is responsible for the atropine-resistant salivary secretion. vasoactive intestinal peptide; substance P; muscarinic block; auriculo-temporal nerve stimulation; fluid, amylase and protein secretion
Address all correspondence to." Dr. J. Ekstr6m, Dept. of Physiology, University of Lund, S61vegatan 10, S-223 62 Lund, Sweden. 0167-0115/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
354
Introduction
Salivary secretion from rat parotid gland is evoked not only by cholinergic and adrenergic agents [1] but also by neuropeptides. Substance P (SP) injected intravenously causes a lively flow of saliva [2]. Vasoactive intestinal peptide (VIP), on the other hand, causes the gland to secrete a small amount of protein-rich saliva [3]. SPand VIP-containing nerve fibres reach the parotid gland via the parasympathetic auriculo-temporal nerve; the total amounts of these peptides in the gland are reduced by about 95% following parasympathetic denervation [4]. Interestingly, even in the presence of atropine and ~- and ~-adrenoceptor blockers electrical stimulation of the auriculo-temporal nerve evokes a secretion of saliva in the rat, which amounts to 5-10% of that obtained upon nerve stimulation before administration of atropine [5]. This atropine-resistant secretion, rich in proteins, is not observed at stimulation frequencies below 5 Hz; the maximal flow rate occurs at 40 Hz [6]. In the present study an attempt was made to deplete the neuronal stores of SP and VIP in the parotid gland by continuous stimulation of the auriculo-temporal nerve. Further, it was investigated whether this protocol would affect the atropine-resistant flow of saliva and output of amylase and proteins.
Methods
48 adult male Sprague-Dawley rats, weighing 303 + 5 g (mean ± S.E.M.) were anaesthetized with chloralose (100 mg/kg) through a femoral venous cannula after induction with diethyl ether. They were provided with a tracheal cannula and a rectal thermometer; the body temperature was kept at about 37.5"C. The duct of the parotid gland was exposed near its entrance in the mouth and cannulated with a glass cannula. The auriculo-temporal nerve, carrying the parasympathetic nerve fibres to the gland [7,8], was exposed medially to the mandible and the nerve was cut where it emerged from the base of the skull. To exclude adrenergic effects the ct-adrenoceptor blocker dihydroergotamine (0.5 mg/kg i.v.) and the fl-adrenoceptor blocker propranolol (0.5 mg/kg i.v.) were given to all the rats. The peripheral end of the nerve was stimulated with supramaximal voltage, 6 V, and at a duration of 2 ms, at a frequency of 40 Hz using a bipolar electrode and a Grass $48 stimulator. Saliva was collected from the cannula in small ice-cooled tubes; the tubes were changed every 10 min. The saliva collected during each 10 min period was weighed and frozen at - 2 0 ° C until analysed for protein and amylase. Protein was determined by the method of Lowry et al. [9] using bovine serum albumin as standard. Amylase was determined colorimetrically using ~-4-nitrophenyl maltoheptaoside (Boehringer Mannheim) as substrate [10]. At the end of the experiment both the stimulated and the non-stimulated parotid gland were removed, weighed, immediately frozen and stored ,at - 70°C until extracted for determination of either VIP or SP. For the assay of VIP-like immunoreactivity (below referred to as VIP), the specimens were extracted in 1-2 ml boiling 0.5 M acetic acid for 10 min, homogenized and centrifuged at 3000 x g for 10 min at room temperature. The supernatants were
355 freeze-dried. The samples were reconstituted in 0.05 M sodium phosphate buffer (pH 7.4) containing 0.5% human serum albumin (Behringwerke, Marburg/Lahn, F.R.G.) and 0.1 M NaC1. Immunoreactive VIP was quantitated using a rabbit antiserum to VIP (No. 7852, Milab, Malmr, Sweden) and radioiodinated VIP [11]; 200 #1 of antiserum (diluted 1 : 16 000) were incubated first with 500/~1 of standard or tissue extract for 3 days at 4"C and then with 200/A (about 4000 cpm) of 125I-VIP for another 2 days at 4°C. Bound and free 12sI-VIP were separated using a second antibody. Each tissue extract was assayed in serial dilution. At times the extracts gave dilution curves that were parallel with the standard VIP curve in the high dilution range only, suggesting the presence of a VIP-like peptide rather than authentic VIP. This was the case when the VIP concentrations in the parotid glands were low. The standard used was synthetic porcine VIP, purchased from Peninsula (Belmont, CA). The concentrations of VIP in the parotid gland extracts were assessed in the high dilution range where the curve was parallel to the standard curve and expressed as equivalents of porcine VIP. It should be recognized that for the reasons specified the absolute values given may be subject to error. For the assay of SP-like immunoreactivity (below referred to as SP), the specimens were minced while still frozen and extracted in 10 volumes of 1.0 M acetic acid in a boiling water bath for 10 min. Following homogenization and centrifugation the supernatants were freeze-dried and redissolved in assay buffer immediately prior to radioimmunoassay. Antiserum K-25 which is directed against the C-terminus of SP was used in a final dilution ot 1:350000, and 12SI-TyrS-Sp served as tracer [12]. The extracts were assayed in duplicate at 3 dilutions. Dilution curves of salivary gland extracts were parallel with the SP standard curve. The levels of VIP and SP were expressed in terms of total amounts, pmol/gland. The drugs used in the studies on secretory responses included atropine sulphate (Sigma), dihydroergotamine methansulphonate (Sandoz), propranolol hydrochloride (ICI) and SP (Sigma). All drugs were injected intravenously. Student's t-test for unpaired or paired data when appropriate was used for estimation of statistical significances. Values are given as means + S.E.M.
Results
Gland weights After continuous stimulation for 20-80 min a periglandular oedema had developed both in the presence and in the absence of atropine. In 35 rats given atropine the wet weight of the parotid glands on the stimulated side was 240 + 6 mg and on the contralateral side 209 + 6 mg (P < 0.001). In 9 animals where the nerve had been stimulated in the absence of atropine the weight of the glands on the stimulated side was 224 q- 9 mg and on the contralateral side 203 + 8 mg (P < 0.01).
Secretory responses There was no spontaneous secretion from the parotid glands. Stimulation of the auriculo-temporal nerve (40 Hz) in non-atropinized animals resulted in salivary flow
356
within 1-2 s. Following treatment with atropine (2 mg/kg i.v.) the secretory response was not apparent until after about 10 s of stimulation. The atropine-resistant secretion during the first 10 min amounted to 231 + 9 mg (n=35) saliva. The output of amylase was 388 + 44 U (n=35) (Fig. 1), and of total protein 881 4- 86 #g (n=35). In the absence of atropine the amount of saliva secreted during the same 10 rain period was 635 + 37 mg (n=9). The output of amylase was 510 + 157 U (n=6) (Fig. 1), and of protein 1215 + 202 gg (n=6). Compared to non-atropinized rats, the amount of saliva, but not that of amylase and protein, secreted during these 10 min was significantly lower (P < 0.001) in atropinized rats. The amounts of saliva, amylase and protein secreted by each non-atropinized and atropinized rat during the initial 10 min period were all set to 100%. The values obtained in the following periods of 10 min were expressed as percentages. In the presence of atropine the flow rate declined markedly with time (Fig. 2). In the second period of observation (10-20 min) the amount of saliva secreted was reduced to 43% (n = 35); it was 13 (n = 26) and 6% (n = 17) in the fourth (30-40 min) and sixth period (50-60 min), respectively. No recovery in secretory responses was noted when stimulation was resumed half an hour after the end of an 80 min period of stimulation. In the absence of atropine the salivary flow rate declined somewhat initially, but remained at a fairly steady level thereafter (Fig. 2). The amount of saliva secreted in the second period was 83% (n = 9); it was 71% (n---5) in the sixth period. Also the output of amylase declined rapidly in the presence of atropine (Fig. 2). It was 28% (n = 35) in the second period, and 6 (n = 19) and 3% (n = 7) in the fourth and sixth period, respectively. In the absence of atropine the decline in amylase output was also great. The figures were 59 (n= 6), 34 (n= 6) and 27% (n= 5) in the second, fourth and sixth period, respectively (Fig. 2). The output of protein in the presence of atropine was 34 + 3% (n= 35) in the second period and 13 + 1% (n = 19) in the fourth period. In the absence of atropine
-ATROPINE +ATROPINE SALIVA AMYLASE 7001 6 600 5001
0 ] ]-
Fig. I. Secretion of saliva (in mg) and output of amylase (in U) from parotid glands during an initial 10-min period of electrical stimulation of the auriculo-temporal nerve (40 Hz, 6 V, 2 ms) in atropinized (2 mg/kg i.v.) and non-atropinized rats. Dihydroergotamine (0.5 mg/kg i.v.) and propranolol (0.5 mg/kg i.v.) were given to all animals. Values are means + S.E.M. Number of observations is indicated.
357 ~ ] -ATROPINE B +ATRORNE SALIVA %100-
4oi
AMYLASE %100
L0666 544
sol B
6o i
e
666
2o I
ol 0
20 40 60 80 min
0
20 40 60 80
min
Fig. 2. Secretion of saliva and output of amylase from parotid glands during 80 min of electrical stimulation of the auriculo-temporal nerve (40 Hz, 6 V, 2 ms) in atropinized and non-atropinized rats. Dihydroergotamine and propranolol were given to all animals (see Fig. 1). Saliva was collected in periods of 10 rain, weighed and analysed. The amounts of saliva and amylase secreted in each rat (both atropinized and non-atropinized) during the first 10-min period were set to 100% (see Fig. 1). All subsequent values were expressed as percentages. Means + S.E.M. Number of observations is indicated.
the protein output was 64 4- 13% (n=6) in the second period and 36 4- 10% (n = 5) in the fourth period. To test the secretory capacity of glands subjected to the prolonged stimulation in the atropinized animals, SP (0.5 #g/kg i.v.) was injected before and 3-5 min after 20-80 min of nerve stimulation. The response to SP after the period of stimulation was found to be larger (by 170%, n--14, P < 0.001) than that obtained before stimulation.
Parotid gland content of VIP and SP The total amount of VIP in 9 non-stimulated parotid glands was 3.4 4- 0.4 pmol, while that of SP in another series of 8 non-stimulated parotid glands was 859 4- 60 pmol. Stimulation of the auriculo-temporal nerve (at 40 Hz, in the presence of atropine) reduced the peptide content. After 20, 40 and 60 min of stimulation the amount of VIP in the stimulated glands, in % of that of contralateral glands, was 63 4- 16 (n = 3), 25 4- 3 (n = 4) and 25 (n = 2; 24, 26), respectively, while the corresponding figures for SP was 56 4- 7 (n = 3), 26 4- 1 (n = 3) and 23 (n = 2; 18, 28).
Discussion
The atropine resistant parotid secretion of fluid and protein, including amylase, decreased to about 5-10% of the initial values within 40 min stimulation of the auriculo-temporal nerve at 40 Hz. The secretion did not cease completely although the stimulation continued for another 40 min. A rest for half an hour was not enough to allow for a recovery in the secretory response. The marked reduction in secretion of water and proteins was not due to a general desensitization of the gland cells, nor was it due to exhaustion of the secretory machinery: the secretory response to SP injected intravenously was greater after than before nerve stimulation and
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further, in rats not given atropine the salivary flow rate and output of protein, and amylase, exceeded that observed in the atropine treated rats. The probable explanation for the rapid decline of the secretory response in the presence of atropine on stimulation of the auriculo-temporal nerve seems to be depletion of non-adrenergic, non-cholinergic transmitter(s). Both VIP and SP would fit into that role, since the parotid gland content of both these peptides was markedly reduced in approximate parallel to the decline in the secretory response. After 40 min of nerve stimulation the total amounts of VIP and SP were reduced to 25%. There was no further decrease during the following 20 min. Interestingly, in the cat dental pulp complete depletion of SP did not occur following long-lasting antidromic nervestimulation [13]. Prolonged nerve stimulation probably evokes a rapid decrease in all neuropeptides in the parotid gland. A depletion can be anticipated since the neuropeptides are synthesized in the cell body of the neurone. Replenishment of the stores in the periphery of the neurone requires axonal transport of preformed, packaged peptides [14]. The present results strongly suggest the involvement of peptides in the regulation of salivary secretion but provide no direct evidence that either SP or VIP is responsible for the atropine-resistant secretion. Since both peptides, injected into the blood stream, stimulate salivary secretion this seems highly likely, however. Interestingly, injection of SP during a period of ongoing stimulation of the auriculo-temporal nerve was recently shown to potentiate the atropine-resistant salivary secretion. Furthermore, the secretory effect of VIP was greatly enhanced by SP (Ekstrfm and Olgart, unpublished observations). Secretion of total protein and amylase seems to a large extent to depend on nonadrenergic, non-cholinergic transmission. In the atropine treated rats total outputs of protein and amylase during the initial 10-min period of nerve stimulation were in both cases 75% of those in untreated rats, whereas the fluid secretion was 35% of controls. These figures are relatively high compared to those earlier reported [5,6]. It is likely that the atropine-resistant secretion has been underestimated previously, since in earlier studies the experimental protocol was to test frequency-response relations before and after atropine in one and the same animal. If the stimulation has been allowed to continue over several minutes at high frequencies the non-adrenergic, non-cholinergic transmitter is probably already partially depleted when the stimulation is repeated in the presence of atropine. Hence, it is probable that atropineresistant salivary secretion will be possible to demonstrate in other species too.
Acknowledgements This study was supported by grants from the Swedish Medical Research Council (14X-05927, 14X-6836, 04X-1007), the Faculties of Medicine and Dentistry, University of Lund, and Dr. P. H~tkansson's Foundation.
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